Low-temperature stress affects reactive oxygen species, osmotic adjustment substances, and antioxidants in rice (Oryza sativa L.) at the reproductive stage

The sensitivity of rice to low-temperature stress (LTS), especially at the reproductive stage, is a primary factor of rice yield fluctuation in cold cultivate region. Here, the changes of reactive oxygen species (ROS), osmotic adjustment substances, and antioxidants in different tissues were analyzed during rice growing under low temperatures (LT) at the reproductive stage. Results showed that LTS increases the levels of proline (Pro), soluble protein (SP), glutathione (GSH), superoxidase (SOD), and ascorbate peroxidase (APX) in LJ25 (LTS-resistant) and LJ11 (LTS-sensitive). The activities of catalase (CAT) and peroxidase (POD) were significantly increased in LJ25 but decreased in LJ11 under LTS, while an opposite trend in ROS and malondialdehyde (MDA) was observed in both varieties. Moreover, most physicochemical properties were higher in flag leaves and panicles compared with those in leaf sheaths. The expression patterns of OsCOIN, OsCATC, OsMAP1, OsPOX1, and OsAPX were the same with phenotypic changes in Pro and the enzymes encoded by them, confirming the accuracy of the physicochemical analysis. Therefore, only CAT and POD increased more in LJ25, suggesting they could be the key factors used for LT-tolerant breeding of rice in cold regions.


Growing condition. The pot experiment was performed in the artificial climate room of Rice Research
Institute of Heilongjiang Academy of Agricultural Sciences (Jiamusi, Heilongjiang, China) in 2019. The cultivation of the experimental materials was according to the methods of Guo et al. 39 with more details. The diameter of the pot was 25 cm, and the height was 23 cm. Before the experiment, each pot was filled with 8 kg of dry paddy soil from the paddy fields of the experimental farm of Rice Research Institute of Heilongjiang Academy of Agricultural Sciences. The basic physical and chemical properties of soil were pH 6.22, organic matter 36.56 g/kg, alkali hydrolysis N, available P, and available K were 105.35 mg/kg, 82.56 mg/kg, and 92.6 mg/kg, respectively. After disinfection, seed soaking, and germination, the seeds of LJ25 and LJ11 were seeded on a seedling plate containing the paddy soil and raised in a plastic shed on April 16. On May 16, when the rice seedlings grew to 3 leaves, and 1 heart stage, the seedlings with the same development process were selected and transplanted into plastic pots. Twenty seedlings with consistent growth were selected and evenly planted in each pot, and each cultivar was planted in twelve pots. Extra tillers of each plant were removed to ensure the consistent growth process of each plant, leaving only the main stem and grown at normal growing condition (28 °C day/22 °C night, 12 h-light/12 h-dark photoperiod, 80% RH). The amounts of fertilization were the same as that of field fertilizer levels in the experimental farm of Rice Research Institute of Heilongjiang Academy of Agricultural Sciences (nitrogen 100 kg/hm 2 , phosphorus 80 kg/hm 2 , and potassium 80 kg/hm 2 ).
LT treatment and sample preparation. Here, the LT treatment and sample preparation were performed following the methods of the cool air treatment indoors 39 . The meiosis at the reproductive stage is known to Scientific Reports | (2022) 12:6224 | https://doi.org/10.1038/s41598-022-10420-8 www.nature.com/scientificreports/ be the most sensitive stage to LTS 40 . However, the determination of the pollen development period through microscopic examination was time-consuming and laborious, although precise. Therefore, the pollen development period was usually estimated based on the auricle distance method. Briefly, the flag leaf 's auricle was approximately 5 cm beneath the of the penultimate leaf 's auricle 40,41 . Till this stage, half of both the cultivars were transferred to another artificial climate room maintained at 12 °C for 4 days (12 h-light/12 h-dark photoperiod, 80% RH) and then returned to the original room till maturity. Next, 0.5 g of fresh flag leaf and 0.5 g of leaf sheath were collected from each group after 0 days, 2 days, and 4 days of treatment at 12 °C, respectively. While, 0.5 g of fresh young spikelets, about 3.5-4.5 mm length, were plucked and collected from the upper third of the panicles after 0 days, 2 days, and 4 days of 12 °C treatment. All the samples of fresh flag leaf, leaf sheath, and fresh young spikelets were immediately frozen in liquid N 2 and stored at − 80 °C. LTS resistance was evaluated on seed setting rates (SSRs) of the main spikelet.
Determination of the content of MDA, Superoxide (O 2 -), and hydrogen peroxide (H 2 O 2 ). The MDA content was determined following the method of Guo et al. 41 . The absorbance of MDA was measured at 440, 532, and 600 nm on UV-Vis Spectrophotometer (Mettler-Toledo UV5Bio, Switzerland). The superoxide (O 2 -) content was determined following the method of Batool et al. 42 . The phosphate buffer (pH 7.8, 0.5 mL), p-aminobenzene sulfonic acid (17 mM, 1 mL), hydroxylammonium chloride (1 mM, 1 mL), and α-naphthylamine (7 mM, 1.0 mL) were mixed, incubated at 25 °C for 60 min and then measured at 530 nm for absorbance. The H 2 O 2 content was measured following the method of Song et al. 19 with minor modifications. Each sample was ground into a homogenate with trichloroacetic acid (0.1%) and then centrifuged at 10,000×g at 4 °C for 20 min. Next, 1 ml of the supernatant was mixed with 2 mL of KI (1 M) and 1 mL of K 2 PO 4 buffer, and the absorbance was measured at 390 nm after 1 h darkness treatment.  39 . The activity of APX (EC 1.11.1.11) was measured following the method of Sato et al. 41 .

Determination of antioxidant enzymes activities.
Determination of the contents of osmotic adjustment substances. Free proline was estimated following the method of Bates et al. with minor modifications 43 . Samples were submerged in 5 mL of sulphosalicylic acid (3%, 100 °C for 10 min); 2 mL of the extract was mixed with ninhydrin reagent containing glacial acetic acid and then incubated for 30 min at 100 °C. After cooling in ice water, 4 mL of toluene was added to the mixture and then measured at 520 nm to determine the proline content. The SP content was estimated via the BCA method following the protocol of Campion et al. 44 . The GSH content was measured according to the method described by Gautam et al. 45 .
Determination of the expression levels of the related genes. OsCOIN, OsCATC , OsMAP1, OsAPXa, and OsPOX1 were selected for determining the expression of different tissues in LJ25 and LJ11 under LTS. Total RNA in all samples was extracted using the respective RNA mini extraction kit (Invitrogen), following the manufacturer's instructions. Primer 3 software was used for designing the primers, The Supplementary Table S1 shows the list of primers. QuantiNova™SYBR®Green PCR kit (Qiagen Inc., Duesseldorf, Germany) was used to perform the qRT-PCR reactions, which was carried out on an ABI StepOne Plus system. The 2 −ΔΔCT method was used for qRT-PCR data analysis with three replicates in each reaction. Actin1 was selected as the reference gene.

Statistical analyses.
All the data obtained were statistically analyzed using the SPSS 19.0 (v20.0, SPSS Inc., Chicago, USA) for variance (ANOVA) analysis. The least significant difference (LSD) test was used to determine the significant differences among treatments (P < 0.05).
Ethics approval and consent to participate. The seeds were kindly provided by the Rice Research Institute of Heilongjiang Academy of Agricultural Sciences, Jiamusi, China. In this study, the experimental research and field studies on plants, including collection of plant material, complied with relevant institutional, national, and international guidelines and legislation.

Results
Effects of LTS on seed setting rates at the reproductive stage. In the present study, we analyzed the SSRs of LJ11 and LJ25 during the LTS after 0 day, 2 days, and 4 days. As shown in Fig. 1, the SSR of LJ11 and LJ25 was 93.3% and 93.7% under normal growing conditions (CK group), respectively, without significant difference between the two cultivars. While, the SSRs in LJ11 significantly decreased to 37.90 % and 8.97% at the 2 and 4 days after LTS (2 DALT and 4 DALT), respectively, suggesting that the spikelets were almost entirely sterile at the end of the LTS. In LJ25, the SSR was only significantly decreased at 4 DALT (59.77%). Moreover, the SSRs in LJ25 after 2 days and 4 days of LTS were both significantly higher than those in LJ11 ( Fig. 1 & Table S2).  Table S3). At 4 DALT, the MDA levels increased significantly by 46.12%, 13.96%, and 28.67% in panicle, flag leaf, and leaf sheath, respectively. While in LJ25, the MDA decreased by 18.07% (P < 0.05), 0.62%, and 4.15% at 2 DALT in panicle, flag leaf, and leaf sheath, respectively, compared to the control group. Moreover, the MDA levels decreased by 15.83% (P < 0.05), 5.81% (P < 0.05), and 4.15% at 4 DALT in panicle, flag leaf, and leaf sheath, respectively. As an indicator of membrane lipid peroxidation, the increase or decrease in MDA levels reflected the degree of cell membrane lipid peroxidation and resistance of plants to stress conditions. The O 2 -content in the panicle of LJ11 significantly increased at 2 and 4 DALT (18.84% and 33.49%, respectively), whereas decreased slightly at 2 and 4 DALT (2.43% and 34.60%, respectively) in LJ25. The response of  Table S3). Moreover, both the MDA and the O 2 levels were significantly higher in LJ11 than in LJ25 at 2 and 4 DALT.

Response
The H 2 O 2 levels in the panicle of LJ11 significantly increased by 90.67% and 155.88% at 2 and 4 DALT, respectively, which was 13.42% and 7.40% at 2 and 4 DALT in LJ25 (not significant). In the flag leaf, the H 2 O 2 levels exhibited the same trends as those in the panicle (105.14% and 175.39% increased at 2 and 4 DALT, respectively), while it was significantly increased by 49.03% and 29.16% at 2 and 4 DALT in LJ25. In the leaf sheath, the response of H 2 O 2 to LTS showed the same pattern as flag leaf, with 96.10% and 136.70% in LJ11, and 36.46% and 24.05% in LJ25 increasing at 2 and 4 DALT, respectively ( Fig. 2g- Table S3). respectively. Lower-case letters represent significant differences among the three treatments for each genotype, upper-case letters represent significant differences between the two genotypes at each treatment. ). In addition, except for the control group in the panicle and the activity at 2 DALT in flag leaf, the activity of POD in LJ25 were all significantly higher than that at the same DALT in LJ11 (Fig. 3a-c & Table S4). The levels of SOD increased significantly by 98.30% and 49.18% at 2 and 4 DALT in LJ11, which were 7.67% and 11.53% in LJ25, respectively. While similar changes occurred in flag leaf between the two cultivars, which were 23.32% and 13.63% in LJ11, and 20.74% and 16.18% in LJ25, respectively. However, in the leaf sheath, the activity of SOD decreased significantly by 20.93% and 30.79% at 2 and 4 DALT in LJ11, while increased significantly by 119.52% and 64.52% in LJ25, respectively. In addition, the activity of SOD was significantly higher in the control group of leaf sheath and 2 DALT of panicle in LJ11 than that in LJ25 (Fig. 3d- Response of several osmotic adjustment substances to LTS at the reproductive stage. As a non-enzymatic antioxidant, the content of Pro in LJ11 only increased at 2 DALT in the panicle, flag leaf, and leaf sheath by 45.32%, 90.43%, and 38.36%, respectively; however, Pro levels increased significantly by 127.15% and represents 'control group. ' '2D' and '4D' represent after 2 days and 4 days of LTS treatment, respectively. Lowercase letters represent significant differences among the three treatments for each genotype; upper-case letters represent significant differences between the two genotypes for each treatment. Data are the means and standard errors of three replicates (n = 3). Data with different letters indicate statistically significant differences among the treatments according to Duncan's multiple range test (P < 0.05).   (d-f) Changes in SOD activities in the panicle, flag leaf, and leaf sheath of LJ11 and LJ25, respectively. (g-i) Changes in CAT activities in the panicle, flag leaf, and leaf sheath of LJ11 and LJ25, respectively. (j-l) Changes in APX activities in the panicle, flag leaf, and leaf sheath of LJ11 and LJ25, respectively. 'CK' represents 'control group. ' '2D' and '4D' represent after 2 days and 4 days of LTS treatment, respectively. Lower-case letters represent significant differences among the three treatments for each genotype; upper-case letters represent significant differences between the two genotypes for each treatment. Data are the means and standard errors of three replicates (n = 3). Data with different letters indicate statistically significant differences among the treatments according to Duncan's multiple range test (P < 0.05).  Table S5).

Discussion
Since rice is grown on the ground and immovable, the type of environmental conditions is crucial for rice survival and development, especially the unfavorable ones, of which LTS is a principal element. As the largest province of rice in north China, Heilongjiang is also an important base of rice commodity grain in China (70% of rice production as a commodity, www. zzys. moa. gov. cn). However, since it is located in the northernmost region of China, LTS, especially on the reproductive stage, seriously restricts the safe production of rice. Thus, LJ25 and respectively. Lower-case letters represent significant differences among the three treatments for each genotype. 'CK' represents 'control group. ' '2D' and '4D' represent after 2 days and 4 days of LTS treatment, respectively. Lower-case letters represent significant differences among the three treatments for each genotype; upper-case letters represent significant differences between the two genotypes for each treatment. Data are the means and standard errors of three replicates (n = 3). Data with different letters indicate statistically significant differences among the treatments according to Duncan's multiple range test (P < 0.05). www.nature.com/scientificreports/ LJ11 were selected for further comprehensive investigation into the metabolic regulatory mechanisms underlying LT tolerance in rice exposed to LTS at the reproductive stage. LJ25 and LJ11 present significantly different LT tolerance at the reproductive stage. LJ25 is one of the strongest LTS resistant crops and is widely cultivated in the third accumulative temperate zone in Heilongjiang Province 5 , representing a strong LT tolerance of rice in the cold region at the reproductive stage. LJ11 with good agronomic and yield characters is weakly resistant to LTS when cultivated in the same temperate region 5 . In addition, LJ25 and LJ11 underwent simultaneous meiosis, ensuring the accuracy of the LTS treatment period and the outcomes. During its evolution, rice has been exposed to a variety of biotic and abiotic stresses and has thus acquired specific physiological adaptations for survival in harsh environments. Antioxidant defense systems play key roles in the alleviation of oxidative damage induced by LTS and include the modulation of osmotic conditions and the coordination of enzymatic and non-enzymatic antioxidants 7,46,47 . Rice produces substantial amounts of ROS under LTS conditions, leading to ROS imbalances that affect the cell's metabolism and damage intracellular proteins, membrane lipids, and DNA 48 . As the first line of defense, SOD is known to detoxify O 2 to form H 2 O 2 and O 2 , while POD, CAT, and APX are the key enzymes that convert H 2 O 2 into H 2 O 49 . In this study, along with the significant increase in the amount of O 2 -observed in all the investigated tissues of LJ11 and LJ25, it was found that SOD activities were also significantly increased, except in the leaf sheath of LJ11. However, as the next step, the activities of POD, CAT, and APX and the concentrations of H 2 O 2 and MDA responded differently in the various samples. The levels of MDA and H 2 O 2 in LJ11 were all significantly increased under LTS while only slightly or even decreasing in LJ25. The POD and CAT levels decreased significantly in LJ11 while increasing remarkably in LJ25. However, the activities of APX in LJ11 and LJ25 showed the same trend, namely, downregulation in all samples in response to LTS. CAT is mainly known to scavenge and break down the H 2 O 2 produced via the fatty acid oxidation photorespiration reaction, reducing the peroxidation of membrane lipids 50 , and POD is known to decompose H 2 O 2 using phenol as the substrate 51 . When exposed to continuous LTS, the cytosolic concentrations of the osmotica (SP and Pro) increased, reducing the water potential and alleviating cellular injury. Proline is known to be positively related to plants' responses to LTS and can be used as an indicator to reflect the LT tolerance of plants and the degree of LTS suffered by plants 52 . In this study, both SP and Pro were found to be significantly increased under LTS in the two cultivars, consistent with the previous reports 53,54 , suggesting that they played positive roles in the physiological response to LTS at the reproductive stage of rice in cold regions. The AsA-GSH cycle, formed by GSH and AsA, is known to be an important route for ROS scavenging, where GSH and AsA reduce ROS directly or act as an enzyme-substrate system to remove ROS 55 . The levels of GSH in both LJ25 and LJ11 were significantly increased after LTS treatments, showing that GSH was also a positive regulator of LTS in rice in cold regions. Moreover, the POD and CAT were significantly upregulated in LJ25 and downregulated in LJ11, suggesting that they played key roles in the response of rice to LTS in cold regions.
Since plants are continuously exposed to the environment, all the tissues are threatened by LTS. The flag leaves are the most important source organs in functional leaves and play a vital role in grain yield 56 . As the main energy storage organs, exposure of rice panicle to LT at the reproductive stage directly causes male sterility and seriously affects rice yield 57 . Leaf-sheaths have different physiological functions in different growth stages of rice, where they could act as flow organs or source and sink organs 58 . Here we investigated the responses of the panicle, flag leaf, and leaf sheath on LJ25 and LJ11 to LTS at the reproductive stage. Previous studies have reported that the antioxidant enzymes of flag leaf increase along with the ROS accumulation to maintain the free radicals at appropriate levels 59,60 . Herein, the amounts of O 2 -, H 2 O 2 , MDA, GSH, APX, Pro, and SP in the panicle, flag leaves, and leaf sheath showed the same changing trends in both LJ11 and LJ25, which were approximately consistent with previous reports. The activities of POD and CAT in all the investigated tissues of LJ11 decreased steadily and significantly with LTS, while significantly increased in LJ25. The activity of SOD in the leaf sheath decreased in LJ11 while increased after 2 days of LTS and slightly decreased after 4 days of LTS in LJ25, consistent with the reports of Xiang et al., who reported that the antioxidant enzymes of leaves could increase under LTS within a certain period and then decline in different degrees 61 .
Previous studies have reported that the accumulation of ROS under LTS could trigger the expression of LTrelated genes accompanied by physiological and biochemical changes 41,62 . OSCOIN, encoding a ring zinc finger protein, is known to be expressed in all the organs of rice and intensively induced by various abiotic stresses, such as LT, ABA, salt, and drought. The overexpression of OsCOIN could upregulate the expression of OsP5CS, enhance the proline content, and significantly improve the tolerance to LT, drought, and salt stresses 63 . In this study, the expression of OsCOIN in most investigate tissues were all upregulated under LTS on the reproductive stage of rice, consistent with previous reports 63 . This result indicated that OsCOIN played a positive role in response to LTS at the reproductive stage of rice in cold regions. OsCATC 47,48,64 , directly encoding catalase, whose expression under LTS was accompanied by the changes in CAT activity, was significantly upregulated in LJ25 while sharply downregulated in LJ11. OsPOX1, as a third peroxidase encoding gene, is known to be expressed in both aboveground and underground tissues and expressed in vessels and anthers when in the early flower stage of microspore development 37,65 . In this study, the expression trends of OsPOX1 were similar to those of OsCATC , indicating that both OsCATC and OsPOX1 could be used for enhancing the LT tolerance of rice in cold regions. Under LTS, ROS accumulation could also promote the signaling network response to LTS, such as the MAPKK-MAPK pathway 66 . OsMAP1, encoding a mitogen-activated protein, is involved in the MAPKK-MAPK pathway. Treatment for 48 h at 12 °C and the ROS accumulation during this time could positively 67 induce OsMAP1 expression; this is consistent with its expression pattern observed in LJ25 in this study, although not in LJ11. These findings indicate that OsMAP1 plays a significant role in LT tolerance in rice in cold regions.

Conclusions
In this study, by analyzing the responses of ROS, osmotic adjustment substances, and antioxidants on different tissues of rice under LTS at the reproductive stage, we derived at the following conclusions: First, the amounts of ROS and MDA were almost significantly increased in LJ11 while slightly increased or significantly decreased in LJ25 under LTS; the contents of those osmotic adjustment substances (Pro, SP, and GSH) were all significantly increased in both LJ11 and LJ25; the antioxidant enzymes, except the CAT and POD (upregulated in LJ25 and downregulated in LJ11), all others showed the same trends in LJ25 and LJ11. Second, most of the contents or activities in leaf sheath were less or weaker than those in flag leave and panicle. Third, the expression of the genes related to the antioxidant enzymes investigated here also showed the same trends with the phenotypic data of those enzymes. To sum up, our findings provided a conceptual model of the effects of LTS on the physiological and biochemical indices and gene expression patterns of rice in cold regions based on our data (Fig. 6) and laid a foundation for rice LT-tolerance breeding in cold regions.

Data availability
The datasets generated during and/or analysed during the current study are available in Supplementary files. www.nature.com/scientificreports/